Kinetic and Spectroscopic Investigation of the Y157F and C93G/Y157F Variants of Cysteine Dioxygenase: Dissecting the Roles of the Second-Sphere Residues C93 and Y157.

In mammals, l-cysteine (Cys) homeostasis is maintained by the mononuclear nonheme iron enzyme cysteine dioxygenase (CDO), which oxidizes Cys to cysteine sulfinic acid. CDO contains a rare post-translational modification, involving the formation of a thioether cross-link between a Cys residue at position 93 (Mus musculus CDO numbering) and a nearby tyrosine at position 157 (Cys-Tyr cross-link). As-isolated CDO contains both the cross-linked and non-cross-linked isoforms, and formation of the Cys-Tyr cross-link during repeated enzyme turnover increases CDO's catalytic efficiency by ∼10-fold. Interestingly, while the C93G CDO variant lacks the Cys-Tyr cross-link, it is similarly active as cross-linked wild-type (WT) CDO. Alternatively, the Y157F CDO variant, which also lacks the cross-link but maintains the free thiolate at position 93, exhibits a drastically reduced catalytic efficiency. These observations suggest that the untethered thiolate moiety of C93 is detrimental to CDO activity and/or that Y157 is essential for catalysis. To further assess the roles of residues C93 and Y157, we performed a spectroscopic and kinetic characterization of Y157F CDO and the newly designed C93G/Y157F CDO variant. Our results provide evidence that the non-cross-linked C93 thiolate stabilizes a water at the sixth coordination site of Cys-bound Y157F Fe(II)CDO. A water is also present, though more weakly coordinated, in Cys-bound C93G/Y157F Fe(II)CDO. The presence of a water molecule, which must be displaced by cosubstrate O2, likely makes a significant contribution to the ∼15-fold and ∼7-fold reduced catalytic efficiencies of the Y157F and C93G/Y157F CDO variants, respectively, relative to cross-linked WT CDO.

TRIM47-CDO1 axis dictates hepatocellular carcinoma progression by modulating ferroptotic cell death through the ubiquitin‒proteasome system.

Hepatocellular carcinoma (HCC) is the predominant form of liver cancer, characterized by high morbidity and mortality rates, as well as unfavorable treatment outcomes. Tripartite motif-containing protein 47 (TRIM47) has been implicated in various diseases including tumor progression with the activity of E3 ubiquitin ligase. However, the precise regulatory mechanisms underlying the involvement of TRIM47 in HCC remain largely unexplored. Here, we provide evidence that TRIM47 exhibits heightened expression in tumor tissues, and its expression is in intimate association with clinical staging and patient prognosis. TRIM47 promotes HCC proliferation, migration, and invasion as an oncogene by in vitro gain- and loss-of-function experiments. TRIM47 knockdown results in HCC ferroptosis induction, primarily through CDO1 involvement to regulate GSH synthesis. Subsequent experiments confirm the interaction between TRIM47 and CDO1 dependent on B30.2 domain, wherein TRIM47 facilitates K48-linked ubiquitination, leading to a decrease in CDO1 protein abundance in HCC. Furthermore, CDO1 is able to counteract the promotional effect of TRIM47 on HCC biological functions. Overall, our research provides novel insight into the mechanism of TRIM47 in CDO1-mediated ferroptosis in HCC cells, highlighting its value as a potential target candidate for HCC therapeutic approaches.

Polymorphisms in the cysteine dioxygenase gene and their association with taurine content in the Pacific oyster Crassostrea gigas.

The Pacific oyster Crassostrea gigas is rich in taurine, which is crucial for its adaptation to the fluctuating intertidal environment and presents significant potential in improving taurine nutrition and boosting immunity in humans. Cysteine dioxygenase (CDO) is a key enzyme involved in the initial step of taurine biosynthesis and plays a crucial role in regulating taurine content in the body. In the present study, polymorphisms of CDO gene in C. gigas (CgCDO) and their association with taurine content were evaluated in 198 individuals. A total of 24 single nucleotide polymorphism (SNP) loci were identified in the exonic region of CgCDO gene by direct sequencing. Among these SNPs, c.279G>A and c.287C>A were found to be significantly associated with taurine content, with the GG and AA genotype at the two loci exhibiting enhanced taurine accumulation (p < 0.05). Haplotype analysis revealed that the 279GG/287AA haplotype had the highest taurine content of 29.24 mg/g, while the 279AA/287CC haplotype showed the lowest taurine content of 21.19 mg/g. These results indicated that the SNPs of CgCDO gene could influence the taurine content in C. gigas and have potential applications in the selective breeding of high-taurine varieties.

Deletion of hepatocyte cysteine dioxygenase type 1, a bile acid repressed gene, enhances glutathione synthesis and ameliorates acetaminophen hepatotoxicity.

Liver is a major organ that metabolizes sulfur amino acids cysteine, which is the substrate for the synthesis of many essential cellular molecules including GSH, taurine, and coenzyme A. Bile acid-activated farnesoid x receptor (FXR) inhibits cysteine dioxygenase type 1 (CDO1), which mediates hepatic cysteine catabolism and taurine synthesis. To define the impact of bile acid inhibition of CDO1 on hepatic sulfur amino acid metabolism and antioxidant capacity, we developed hepatocyte-specific CDO1 knockout mice (Hep-CDO1 KO) and hepatocyte specific CDO1 transgenic mice (Hep-CDO1 Tg). Liver metabolomics revealed that genetic deletion of hepatic CDO1 reduced de novo taurine synthesis but had no impact on hepatic taurine abundance or bile acid conjugation. Consistent with reduced cysteine catabolism, Hep-CDO1 KO mice showed increased hepatic cysteine abundance but unaltered methionine cycle intermediates and coenzyme A synthesis. Upon acetaminophen overdose, Hep-CDO1 KO mice showed increased GSH synthesis capacity and alleviated liver injury. In contrast, hepatic CDO1 overexpression in Hep-CDO1 Tg mice stimulated hepatic cysteine to taurine conversion, resulting in reduced hepatic cysteine abundance. However, Hep-CDO1 Tg mice and WT showed similar susceptibility to acetaminophen-induced liver injury. Hep-CDO1 Tg mice showed similar hepatic taurine and coenzyme A compared to WT mice. In summary, these findings suggest that bile acid and FXR signaling inhibition of CDO1-mediated hepatic cysteine catabolism preferentially modulates hepatic GSH synthesis capacity and antioxidant defense, but has minimal effect on hepatic taurine and coenzyme A abundance. Repression of hepatic CDO1 may contribute to the hepatoprotective effects of FXR activation under certain pathologic conditions.

Hypoxia-inducible factor induces cysteine dioxygenase and promotes cysteine homeostasis in Caenorhabditis elegans.

Dedicated genetic pathways regulate cysteine homeostasis. For example, high levels of cysteine activate cysteine dioxygenase, a key enzyme in cysteine catabolism in most animal and many fungal species. The mechanism by which cysteine dioxygenase is regulated is largely unknown. In an unbiased genetic screen for mutations that activate cysteine dioxygenase (cdo-1) in the nematode Caenorhabditis elegans, we isolated loss-of-function mutations in rhy-1 and egl-9, which encode proteins that negatively regulate the stability or activity of the oxygen-sensing hypoxia inducible transcription factor (hif-1). EGL-9 and HIF-1 are core members of the conserved eukaryotic hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 is largely independent of EGL-9 prolyl hydroxylase activity and the von Hippel-Lindau E3 ubiquitin ligase, the classical hypoxia signaling pathway components. We demonstrate that C. elegans cdo-1 is transcriptionally activated by high levels of cysteine and hif-1. hif-1-dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is also sufficient to drive sulfur amino acid metabolism. Thus, the regulation of cdo-1 by hif-1 reveals a negative feedback loop that maintains cysteine homeostasis. High levels of cysteine stimulate the production of an H2S signal. H2S then acts through the rhy-1/cysl-1/egl-9 signaling pathway to increase HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.

Proteins are large molecules in our cells that perform various roles, from acting as channels through which nutrients can enter the cell, to forming structural assemblies that help the cell keep its shape. Proteins are formed of chains of building blocks called amino acids. There are 20 common amino acids, each with a different 'side chain' that confers it with specific features. Cysteine is one of these 20 amino acids. Its side chain has a 'thiol' group, made up of a sulfur atom and a hydrogen atom. This thiol group is very reactive, and it is an essential building block of enzymes (proteins that speed up chemical reactions within the cell), structural proteins and signaling molecules. While cysteine is an essential amino acid for the cell to function, excess cysteine can be toxic. The concentration of cysteine in animal cells is tightly regulated by an enzyme called cysteine dioxygenase. This enzyme is implicated in two rare conditions that affect metabolism, where the product of cysteine dioxygenase is a key driver of disease severity. Additionally, cysteine dioxygenase acts as a tumor suppressor gene, and its activity becomes blocked in diverse cancers. Understanding how cysteine dioxygenase is regulated may be important for research into these conditions. While it has been shown that excess cysteine drives the production and activity of cysteine dioxygenase, how the cell detects high levels of cysteine remained unknown. Warnhoff et al. sought to resolve this question using the roundworm Caenorhabditis elegans. First, the scientists demonstrated that, like in mammals, high levels of cysteine drive the production of cysteine dioxygenase in C. elegans. Next, the researchers used an approach called an unbiased genetic screening to find genes that induce cysteine dioxygenase production when they are mutated. These experiments revealed that the protein HIF-1 can drive the production of cysteine dioxygenase when it is activated by a pathway that senses hydrogen sulfide gas. Based on these results, Warnhoff et al. propose that high levels of cysteine lead to the production of hydrogen sulfide gas that in turn drives the production of cysteine dioxygenase via HIF-1 activation of gene expression. The results reported by Warnhoff et al. suggest that modulating HIF-1 signaling could control the activity of cysteine dioxygenase. This information could be used in the future to develop therapies for molybdenum cofactor deficiency, isolated sulfite oxidase deficiency and several types of cancer. However, first it will be necessary to demonstrate that the same signaling pathway is active in humans.

Targeted demethylation of the CDO1 promoter based on CRISPR system inhibits the malignant potential of breast cancer cells.

BACKGROUND: Cysteine dioxygenase 1 (CDO1) is frequently methylated, and its expression is decreased in many human cancers including breast cancer (BC). However, the functional and mechanistic aspects of CDO1 inactivation in BC are poorly understood, and the diagnostic significance of serum CDO1 methylation remains unclear. METHODS: We performed bioinformatics analysis of publicly available databases and employed MassARRAY EpiTYPER methylation sequencing technology to identify differentially methylated sites in the CDO1 promoter of BC tissues compared to normal adjacent tissues (NATs). Subsequently, we developed a MethyLight assay using specific primers and probes for these CpG sites to detect the percentage of methylated reference (PMR) of the CDO1 promoter. Furthermore, both LentiCRISPR/dCas9-Tet1CD-based CDO1-targeted demethylation system and CDO1 overexpression strategy were utilized to detect the function and underlying mechanism of CDO1 in BC. Finally, the early diagnostic value of CDO1 as a methylation biomarker in BC serum was evaluated. RESULTS: CDO1 promoter was hypermethylated in BC tissues, which was related to poor prognosis (p < .05). The CRISPR/dCas9-based targeted demethylation system significantly reduced the PMR of CDO1 promotor and increased CDO1 expression in BC cells. Consequently, this leads to suppression of cell proliferation, migration and invasion. Additionally, we found that CDO1 exerted a tumour suppressor effect by inhibiting the cell cycle, promoting cell apoptosis and ferroptosis. Furthermore, we employed the MethyLight to detect CDO1 PMR in BC serum, and we discovered that serum CDO1 methylation was an effective non-invasive biomarker for early diagnosis of BC. CONCLUSIONS: CDO1 is hypermethylated and acts as a tumour suppressor gene in BC. Epigenetic editing of abnormal CDO1 methylation could have a crucial role in the clinical treatment and prognosis of BC. Additionally, serum CDO1 methylation holds promise as a valuable biomarker for the early diagnosis and management of BC.

Genome-Wide Identification and Analysis of the Plant Cysteine Oxidase (PCO) Gene Family in Brassica napus and Its Role in Abiotic Stress Response.

Plant Cysteine Oxidase (PCO) is a plant O2-sensing enzyme catalyzing the oxidation of cysteine to Cys-sulfinic acid at the N-termini of target proteins. To better understand the Brassica napus PCO gene family, PCO genes in B. napus and related species were analyzed. In this study, 20, 7 and 8 PCO genes were identified in Brassica napus, Brassica rapa and Brassica oleracea, respectively. According to phylogenetic analysis, the PCOs were divided into five groups: PCO1, PCO2, PCO3, PCO4 and PCO5. Gene organization and motif distribution analysis suggested that the PCO gene family was relatively conserved during evolution. According to the public expression data, PCO genes were expressed in different tissues at different developmental stages. Moreover, qRT-PCR data showed that most of the Bna/Bra/BoPCO5 members were expressed in leaves, roots, flowers and siliques, suggesting an important role in both vegetative and reproductive development. Expression of BnaPCO was induced by various abiotic stress, especially waterlogging stress, which was consistent with the result of cis-element analysis. In this study, the PCO gene family of Brassicaceae was analyzed for the first time, which contributes to a comprehensive understanding of the origin and evolution of PCO genes in Brassicaceae and the function of BnaPCO in abiotic stress responses.

A Single DNA Point Mutation Leads to the Formation of a Cysteine-Tyrosine Crosslink in the Cysteine Dioxygenase from Bacillus subtilis.

Cysteine dioxygenase (CDO) is a non-heme iron-containing enzyme that catalyzes the oxidation of cysteine (Cys) to cysteine sulfinic acid (CSA). Crystal structures of eukaryotic CDOs revealed the presence of an unusual crosslink between the sulfur of a cysteine residue (C93 in Mus musculus CDO, MmCDO) and a carbon atom adjacent to the phenyl group of a tyrosine residue (Y157). Formation of this crosslink occurs over time as a byproduct of catalysis and increases the catalytic efficiency of CDO by at least 10-fold. Interestingly, in bacterial CDOs, the residue corresponding to C93 is replaced by a highly conserved glycine (G82 in Bacillus subtilis CDO, BsCDO), which precludes the formation of a C-Y crosslink in these enzymes; yet bacterial CDOs achieve turnover rates paralleling those of fully crosslinked eukaryotic CDOs. In the present study, we prepared the G82C variant of BsCDO to determine if a single DNA point mutation could lead to C-Y crosslink formation in this enzyme. We used gel electrophoresis, peptide mass spectrometry, electron paramagnetic resonance spectroscopy, and kinetic assays to characterize this variant alongside the natively crosslinked wild-type (WT) MmCDO and the natively non-crosslinked WT BsCDO. Collectively, our results provide compelling evidence that the G82C BsCDO variant is indeed capable of C-Y crosslink formation. Our kinetic studies indicate that G82C BsCDO has a reduced catalytic efficiency compared to WT BsCDO and that activity increases as the ratio of crosslinked to non-crosslinked enzyme increases. Finally, by carrying out a bioinformatic analysis of the CDO family, we were able to identify a large number of putatively crosslinked bacterial CDOs, the majority of which are from Gram-negative pathogenic bacteria.

Testosterone enhances taurine synthesis by upregulating androgen receptor and cysteine sulfinic acid decarboxylase expressions in male mouse liver.

Taurine is an end-product of cysteine metabolism, whereas cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSAD) are key enzymes regulating taurine synthesis. Sex steroids, including estrogens and androgens, are associated with liver physiopathological processes; however, we still do not know whether taurine and sex steroids interact in regulating liver physiology and hepatic diseases, and whether there are sex differences, although our recent study shows that the estrogen is involved in regulating taurine synthesis in mouse liver. The present study was thus proposed to identify whether 17-ß-estradiol and testosterone (T) play their roles by regulating CDO and CSAD expression and taurine synthesis in male mouse liver. Our results demonstrated that testosterone did not have a significant influence on CDO expression but significantly enhanced CSAD, androgen receptor (AR) expressions, and taurine levels in mouse liver, cultured hepatocytes, and HepG2 cells, whereas these effects were abrogated by AR antagonist flutamide. Furthermore, our results showed that testosterone increased CSAD-promoter-luciferase activity through the direct interaction of the AR DNA binding domain with the CSAD promoter. These findings first demonstrate that testosterone acts as an important factor to regulate sulfur amino acid metabolism and taurine synthesis through AR/CSAD signaling pathway. In addition, the in vivo and in vitro experiments showed that 17-ß-estradiol has no significant effects on liver CSAD expression and taurine synthesis in male mice and suggest that the effects of sex steroids on the taurine synthesis in mouse liver have sex differences. These results are crucial for understanding the physiological functions of taurine/androgen and their interacting mechanisms in the liver.NEW & NOTEWORTHY This study demonstrates that testosterone functions to enhance taurine synthesis by interacting with androgen receptor and binding to cysteine sulfinate decarboxylase (CSAD) promoter zone. Whereas estrogen has no significant effects either on liver CSAD expression or taurine synthesis in male mice and suggests that the effects of sex steroids on taurine synthesis in the liver have gender differences. These new findings are the potential for establishing effective protective and therapeutic strategies for liver diseases.

Adipose tissue cysteine dioxygenase type 1 is associated with an anti-inflammatory profile, impacting on systemic metabolic traits.

BACKGROUND: Adipose tissue is a source of multiple factors that modulate systemic insulin sensitivity and cardiovascular risk. Taurine is obtained from the diet but it is less known that it is endogenously synthesized by cysteine dioxygenase type 1 (CDO1). CDO1 exerts a role in adipose tissue from rodent models, but the potential translational value in humans is not available in the literature. METHODS: CDO1 gene expression was analysed in visceral and subcutaneous adipose tissue samples in association with metabolic traits in participants with different degrees of obesity in four independent cohorts. CDO1 was also evaluated in isolated human adipocytes in vitro. Mechanistically, CDO1gene knockdown (KD) of human preadipocytes and adipose-derived mesenchymal stem cells (ASC52telo) (using lentiviral particles) was also evaluated. Mitochondrial respiratory function of adipocytes was evaluated using Seahorse. FINDINGS: Both visceral (VAT) and subcutaneous adipose tissue (SAT) CDO1 mRNA was associated with gene expression markers of adipose tissue function in the four cohorts. Higher CDO1 expression was linked to decreased fasting triglycerides and blood HbA1c even after adjusting by age, BMI and sex. In addition, CDO1 mRNA positively correlated with the expression of genes involved in adipogenesis and negatively with different inflammatory markers. Both VAT and SAT CDO1 mRNA was mainly expressed in adipocytes and significantly increased during adipocyte differentiation, but attenuated under inflammatory conditions. Mechanistically, CDO1 gene KD reduced taurine biosynthesis, evidencing lower CDO1 activity. In both human preadipocytes and ASC52telo cells, CDO1 gene KD resulted in decreased gene expression markers of adipogenesis (ADIPOQ, FABP4, FASN, SLC2A4, CEBPA) and increased inflammatory genes (TNF and IL6) during adipocyte differentiation. Of note, CDO1 gene KD led to decreased mitochondrial respiratory function in parallel to decreased expression of mitochondrial function-, but not biogenesis-related genes. INTERPRETATION: Current findings show the relevance of CDO1 in adipose tissue physiology, suggesting its contribution to an improved systemic metabolic profile. FUNDING: This work was partially supported by research grants PI16/01173, PI19/01712, PI20/01090 and PI21/01361 from the Instituto de Salud Carlos III from Spain, Fondo Europeo de Desarrollo Regional (FEDER) funds, and VII Spanish Diabetes Association grants to Basic Diabetes Research Projects led by young researchers.

Emerging roles for thiol dioxygenases as oxygen sensors.

Cysteine dioxygenases, 3-mercaptopropionate dioxygenases and mercaptosuccinate dioxygenases are all thiol dioxygenases (TDOs) that catalyse oxidation of thiol molecules to sulphinates. They are Fe(II)-dependent dioxygenases with a cupin fold that supports a 3xHis metal-coordinating triad at the active site. They also have other, broadly common features including arginine residues involved in substrate carboxylate binding and a conserved trio of residues at the active site featuring a tyrosine important in substrate binding catalysis. Recently, N-terminal cysteinyl dioxygenase enzymes (NCOs) have been identified in plants (plant cysteine oxidases, PCOs), while human 2-aminoethanethiol dioxygenase (ADO) has been shown to act as both an NCO and a small molecule TDO. Although the cupin fold and 3xHis Fe(II)-binding triad seen in the small molecule TDOs are conserved in NCOs, other active site features and aspects of the overall protein architecture are quite different. Furthermore, the PCOs and ADO appear to act as biological O2 sensors, as shown by kinetic analyses and hypoxic regulation of the stability of their biological targets (N-terminal cysteine oxidation triggers protein degradation via the N-degron pathway). Here, we discuss the emergence of these two subclasses of TDO including structural features that could dictate their ability to bind small molecule or polypeptide substrates. These structural features may also underpin the O2 -sensing capability of the NCOs. Understanding how these enzymes interact with their substrates, including O2 , could reveal strategies to manipulate their activity, relevant to hypoxic disease states and plant adaptive responses to flooding.

Targeting plant cysteine oxidase activity for improved submergence tolerance.

Plant cysteine oxidases (PCOs) are plant O2 -sensing enzymes. They catalyse the O2 -dependent step which initiates the proteasomal degradation of Group VII ethylene response transcription factors (ERF-VIIs) via the N-degron pathway. When submerged, plants experience a reduction in O2 availability; PCO activity therefore decreases and the consequent ERF-VII stabilisation leads to upregulation of hypoxia-responsive genes which enable adaptation to low O2 conditions. Resulting adaptations include entering an anaerobic quiescent state to maintain energy reserves and rapid growth to escape floodwater and allow O2 transport to submerged tissues. Stabilisation of ERF-VIIs has been linked to improved survival post-submergence in Arabidopsis, rice (Oryza sativa) and barley (Hordeum vulgare). Due to climate change and increasing flooding events, there is an interest in manipulating the PCO/ERF-VII interaction as a method of improving yields in flood-intolerant crops. An effective way of achieving this may be through PCO inhibition; however, complete ablation of PCO activity is detrimental to growth and phenotype, likely due to other PCO-mediated roles. Targeting PCOs will therefore require either temporary chemical inhibition or careful engineering of the enzyme structure to manipulate their O2 sensitivity and/or substrate specificity. Sufficient PCO structural and functional information should make this possible, given the potential to engineer site-directed mutagenesis in vivo using CRISPR-mediated base editing. Here, we discuss the knowledge still required for rational manipulation of PCOs to achieve ERF-VII stabilisation without a yield penalty. We also take inspiration from the biocatalysis field to consider how enzyme engineering could be accelerated as a wider strategy to improve plant stress tolerance and productivity.

Prospective study to validate the clinical utility of DNA diagnosis of peritoneal fluid cytology test in gastric cancer.

The clinical efficacy of DNA cytology test (CY) in gastric cancer (GC) has been retrospectively proposed using cancer-specific methylation of cysteine dioxygenase type 1 (CDO1). We confirmed the clinical utility of DNA CY in a prospective cohort. Four hundred GC samples were prospectively collected for washing cytology (UMIN000026191), and detection of the DNA methylation of CDO1 was assessed by quantitative methylation-specific PCR in the sediments. Endpoint was defined as the match rate between conventional CY1 and DNA CY1 (diagnostic sensitivity), and the DNA CY0 rate (diagnostic specificity) in pStage IA. DNA CY1 was detected in 45 cases (12.5%), while CY1 was seen in 31 cases (8.6%) of 361 chemotherapy-naïve samples, where the sensitivity and specificity of the DNA CY in the peritoneal solutions were 74.2% and 96.5%, respectively. The DNA CY was positive for 3.5/0/4.9/11.4/58.8% in pStage IA/IB/II/III/IV, respectively (P < .01). In the multivariate analysis, DNA CY1 was independently correlated with pathological tumor depth (pT) (P = .0012), female gender (P = .0099), CY1 (P = .0135), P1 (P = .019), and carcinoembryonic antigen (CEA) (P = .036). The combination of DNA CY1 and P factor nearly all covered the potential peritoneal dissemination (P1 and/or CY1 and/or DNA CY1) (58/61:95.1%). DNA CY1 had a significantly poorer prognosis than DNA CY0 in GC patients (P < .0001). DNA CY1 detected by CDO1 promoter DNA methylation has a great value to detect minimal residual disease of the peritoneum in GC clinics, representing poor prognosis as a novel single DNA marker.

1H NMR metabolomic and transcriptomic analyses reveal urinary metabolites as biomarker candidates in response to protein undernutrition in adult rats.

Protein undernutrition contributes to the development of various diseases in broad generations. Urinary metabolites may serve as non-invasive biomarkers of protein undernutrition; however, this requires further investigation. We aimed to identify novel urinary metabolites as biomarker candidates responsive to protein undernutrition. Adult rats were fed control (CT; 14 % casein) or isoenergetic low-protein (LP; 5 % casein) diets for 4 weeks. 1H NMR metabolomics was applied to urine, plasma and liver samples to identify metabolites responsive to protein undernutrition. Liver samples were subjected to mRNA microarray and quantitative PCR analyses to elucidate the mechanisms causing fluctuations in identified metabolites. Urinary taurine levels were significantly lower in the LP group than in the CT group at week 1 and remained constant until week 4. Hepatic taurine level and gene expression level of cysteine dioxygenase type 1 were also significantly lower in the LP group than in the CT group. Urinary trimethylamine N-oxide (TMAO) levels were significantly higher in the LP group than in the CT group at week 2 and remained constant until week 4. Hepatic TMAO level and gene expression levels of flavin-containing mono-oxygenase 1 and 5 were also significantly higher in the LP group than in the CT group. In conclusion, urinary taurine and TMAO levels substantially responded to protein undernutrition. Furthermore, changes in hepatic levels of these metabolites and gene expressions associated with their metabolic pathways were also reflected in their fluctuating urinary levels. Thus, taurine and TMAO could act as non-invasive urinary biomarker candidates to detect protein undernutrition.

Prediction of Efficacy of Postoperative Chemotherapy by DNA Methylation of CDO1 in Gastric Cancer.

BACKGROUND: CDO1 is a presumed tumor suppressor gene in human cancers, the expression of which is silenced by promoter DNA methylation. Moreover, CDO1 harbors functionally oncogenic aspects through modification of mitochondrial membrane potential. We recently proposed that this oncogenic feature allows for the prediction of the efficacy of postoperative chemotherapy in colon cancer. The present study aims to elucidate the efficacy of prediction of success of postoperative chemotherapy in advanced gastric cancer to improve the treatment strategy of patients. MATERIALS AND METHODS: Forced expression of CDO1 in gastric cancer cell lines was assessed using the JC-1 assay. Promoter DNA methylation was investigated in quantitative TaqMan methylation-specific polymerase chain reaction in 321 pathological stage II/III advanced gastric cancer cases treated by curative gastrectomy with or without postoperative chemotherapy. RESULTS: (1) Forced expression of CDO1 led to increased mitochondrial membrane potential, accompanied by augmented survival in gastric cancer cells under anaerobic conditions. These results suggest that CDO1-expressing cancer cells survive more easily in anaerobic lesions which are inaccessible to anticancer drugs. (2) Intriguingly, in cases with the highest CDO1 methylation (ranging from 15% to 40%), patients with postoperative chemotherapy showed significantly better survival than those with no postoperative chemotherapy. (3) A robust prognostic difference was observed that was explained by differential recurrences of distant metastasis (P = 0.0031), followed by lymph node (P = 0.0142) and peritoneal dissemination (P = 0.0472). CONCLUSIONS: The oncogenic aspects of CDO1 can be of use to determine patients with gastric cancer who will likely respond to treatment of invisible systemic dissemination by postoperative adjuvant chemotherapy.

Detection of Promoter DNA Methylation in Urine and Plasma Aids the Detection of Non-Small Cell Lung Cancer.

PURPOSE: Low-dose CT screening can reduce lung cancer-related mortality. However, CT screening has an FDR of nearly 96%. We sought to assess whether urine samples can be a source for DNA methylation-based detection of non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: This nested case-control study of subjects with suspicious nodules on CT imaging obtained plasma and urine samples preoperatively. Cases (n = 74) had pathologic confirmation of NSCLC. Controls (n = 27) had a noncancer diagnosis. We detected promoter methylation in plasma and urine samples using methylation on beads and quantitative methylation-specific real-time PCR for cancer-specific genes (CDO1, TAC1, HOXA7, HOXA9, SOX17, and ZFP42). RESULTS: DNA methylation at cancer-specific loci was detected in both plasma and urine, and was more frequent in patients with cancer compared with controls for all six genes in plasma and in CDO1, TAC1, HOXA9, and SOX17 in urine. Univariate and multivariate logistic regression analysis showed that methylation detection in each one of six genes in plasma and CDO1, TAC1, HOXA9, and SOX17 in urine were significantly associated with the diagnosis of NSCLC, independent of age, race, and smoking pack-years. When methylation was detected for three or more genes in both plasma and urine, the sensitivity and specificity for lung cancer diagnosis were 73% and 92%, respectively. CONCLUSIONS: DNA methylation-based biomarkers in plasma and urine could be useful as an adjunct to CT screening to guide decision-making regarding further invasive procedures in patients with pulmonary nodules.

[The Role of Plasma CDO1 Methylation in the Early Diagnosis of Lung Cancer].

BACKGROUND: The incidence and mortality of lung cancer often rank first in all malignant tumors. DNA methylation, as one of epigenetics, often participates in the development and progression of tumors. CDO1 as a tumor suppressor gene always undergoes methylation changes early in tumor development. Therefore, this study aims to discuss the value of CDO1 methylation in the early diagnosis of lung cancer. METHODS: Peripheral blood samples were collected from tumor patients and healthy people. Detection of the methylation level of CDO1 in plasma by sulfite modification and quantitative real-time PCR. RESULTS: The level of gene methylation in peripheral blood of lung cancer patients was significantly higher than that of benign lung disease patients and healthy people. The methylation level of CDO1 was significantly different in the stratified comparison of gender, lymph node metastasis and tumor-node-metastasis (TNM) stage (P<0.05). The sensitivity and specificity of CDO1 were 52.2% and 78.6%, respectively. The overall accuracy of the diagnosis was significantly higher than that of the clinical tumor markers, and the sensitivity of CDO1 to stage I and II patients was the highest (40.8%, 47.1%). In addition, CDO1 could effectively increase the sensitivity of diagnosis in multiple joint examinations. CONCLUSIONS: Detecting the methylation level of CDO1 has a potentially huge advantage for the early diagnosis of lung cancer.

Promoter DNA Hypermethylation of the Cysteine Dioxygenase 1 (CDO1) Gene in Intraductal Papillary Mucinous Neoplasm (IPMN).

BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) involves adenoma (IPMA), a precancerous lesion, cancer (IPMC) including high-grade dysplasia (HGD), and invasive carcinoma (IC). DNA markers of IPMN are required for detection of invasive disease, and cysteine dioxygenase 1 (CDO1) gene promoter hypermethylation is a potential candidate. However, it has never been investigated in the context of IPMN. PATIENTS AND METHODS: A total of 107 IPMN tumor tissues, including 41 IPMC and 66 IPMA, were studied. CDO1 promoter methylation was quantified using TaqMan quantitative methylation-specific polymerase chain reaction (qMSP) in patients with IPMN and other pancreatic cystic disorders after pancreatectomy. RESULTS: The methylation values (TaqMeth Vs) of CDO1 increased when noncancerous pancreas tissues were compared with IPMA and HGD (p < 0.0001). Among IPMC, the TaqMeth Vs in IC were not significantly higher than in HGD. The TaqMeth Vs of the solid tumors were higher than those of the cystic tumors (p = 0.0016), which were in turn higher than the corresponding noncancerous tissues (p < 0.0001). Prognostic analysis revealed that high TaqMeth Vs (≥ 14.1) resulted in a poorer prognosis than low TaqMeth Vs (< 14.1) (p < 0.0001). In other pancreatic cystic diseases, only malignant mucinous cystic neoplasm showed DNA hypermethylation of its promoter. A pilot study in pancreatic juice confirmed methylation in all IPMN samples but not in benign pancreatic diseases (p = 0.0277). CONCLUSIONS: CDO1 promoter hypermethylation is extremely specific to IPMN and may accumulate with IPMN tumor progression during the adenoma-carcinoma sequence. It might be a promising candidate as a diagnostic marker of pancreatic cystic diseases.

Diagnostic potential of hypermethylation of the cysteine dioxygenase 1 gene (CDO1) promoter DNA in pancreatic cancer.

DNA markers for pancreatic ductal adenocarcinoma (PDAC) are urgently needed for detection of minimally invasive disease. The epigenetic relevance of the cysteine dioxygenase 1 gene (CDO1) has been never investigated in PDAC. Three studies, including cellular experiments, tissue validation, and pilot testing for pancreatic cytology, were carried out. Promoter DNA methylation value (MV) of CDO1 was quantified by quantitative methylation-specific PCR. CDO1 expression was consistent with its promoter DNA methylation in 7 PDAC cell lines. In 160 retrospectively collected primary PDAC tumor tissues, MV was significantly higher compared to the corresponding noncancerous pancreas (area under the receiver operating characteristic curve [AUC] = 0.97, P < .0001), and CDO1 hypermethylation was highly specific to PDAC tumor tissues. CDO1 hypermethylation group (MV over 19) was significantly associated with diverse prognostic factors in PDAC. Surprisingly, it was significantly higher in prospectively collected PDAC cytology samples (n = 37), including both pancreatic juice (n = 12) and endoscopic ultrasound-fine needle aspiration (EUS-FNA) cytology (n = 25) compared to pancreatic benign diseases (AUC = 0.96, P < .0001). Detection of PDAC was confirmed by DNA testing in 35 of 37 patients (95% sensitivity); thus, it was more sensitive than cytology (33%) or EUS-FNA cytology (88%). Promoter DNA methylation of CDO1 is extremely specific for PDAC tumors, and accumulates with PDAC tumor progression. It could be a definitive diagnostic marker of PDAC in pancreatic juice or EUS-FNA cytology.

Highly Efficient Real-Time Droplet Analysis Platform for High-Throughput Interrogation of DNA Sequences by Melt.

Droplet microfluidic platforms have greatly enhanced the throughput and sensitivity of single-molecule and single-cell analyses. However, real-time analyses of individual droplets remain challenging. Most droplet microfluidic platforms have fundamental drawbacks that undermine their utility toward applications that rely on real-time monitoring to identify rare variants, such as bacterial persistence, drug discovery, antibody production, epigenetic biomarker analyses, etc. We present a platform for high-density droplet trapping and real-time analysis with 100% loading and trapping efficiency at a packing density of 110,000 droplets per in2. To demonstrate real-time analysis capabilities, we perform digital PCR and parallelized digital high-resolution melt curve acquisition on droplets to discriminate methylation levels of a tumor suppressor gene, CDO1, on a molecule-by-molecule basis. We hope that this platform, which is compatible with a large range of droplet sizes and generation technologies, may facilitate high-throughput real-time analyses on a molecule-by-molecule or cell-by-cell basis of heterogeneous populations.